US6096710A - Collagen-like peptoid residue-containing structures - Google Patents

Collagen-like peptoid residue-containing structures Download PDF

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US6096710A
US6096710A US08/668,380 US66838096A US6096710A US 6096710 A US6096710 A US 6096710A US 66838096 A US66838096 A US 66838096A US 6096710 A US6096710 A US 6096710A
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gly
pro
collagen
nleu
peptoid
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Murray Goodman
Joseph P. Taulane
Yangbo Feng
Giuseppe Melacini
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University of California
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University of California
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Assigned to REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE reassignment REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FENG, YANGBO, GOODMAN, MURRAY, MELACINI, GIUSEPPE, TAULANE, JOSEPH P.
Priority to CA002237845A priority patent/CA2237845A1/en
Priority to JP9519839A priority patent/JP2000500497A/ja
Priority to AU10549/97A priority patent/AU716531B2/en
Priority to PCT/US1996/018521 priority patent/WO1997019106A2/en
Priority to EP96941391A priority patent/EP0861264A2/en
Priority to US09/388,916 priority patent/US6329506B1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention relates to synthetic collagen-like polymers comprising repeating trimeric amino acid sequences or trimeric sequences wherein one of the amino acids has been replaced by a peptoid residue. It relates specifically to synthetic collagen polymers having a collagen-like triple helical conformation which consists of three polyproline II-like chains. It also relates to the use of template molecules to induce formation of a helical array.
  • Native collagen has a primary structure of repeating trimeric amino acid sequences. Within a helical region, which constitutes about 95% of the molecule, the amino acid glycine (Gly) occurs at every third position of a peptide trimer. Imino residues (I), either proline (Pro) and hydroxyproline (Hyp), occur in 56% of the trimers, 20% as Gly-X-I; 27% as Gly-I-Y; and 9% as Gly-I-I. Pro usually occurs in the second position in the repeating trimer; while Hyp usually occurs in the last position. (Bhatnagar, R. and R. Rapaka (1976) Chap. 10 in Biochemistry of Collagen R. Ramachandren, ed. Plenum Press, New York. pp.481-482).
  • Tripeptide sequences wherein X and Y are amino acid residues other than proline (Pro) or hydroxyproline (Hyp) make up 44% of the collagen amino acid trimers.
  • Glutamic acid, leucine, and phenylalanine occur mostly in the X position and threonine, glutamine, methionine, arginine and lysine occur mostly at the Y position.
  • glutamine, methionine, arginine and lysine occur mostly at the Y position.
  • the X position amino acids have bulky side chains.
  • Synthetic collagens are of interest because they provide materials for collagen-like biomaterials having diverse clinical applications, including use in drug delivery devices, ocular devices, and wound healing materials. Because the proline and hydroxyproline (a post-translationally modified proline residue) residues are abundant in natural collagen sequences, many sequential polymers composed of the trimeric amino acid sequences Gly-Pro-Xaa and Gly-Xaa-Pro (where Xaa is any natural amino acid residue) have been prepared to mimic the collagen structures. (Segal, D. M., and Traub, W.(1969) J. Mol. Biol. 43:487-496 disclose poly(L-alanyl-L-prolyl-glycine); Segal, D. (1969) J. Mol. Biol.
  • Collagen has a characteristic tertiary, secondary and primary structure. Most polypeptides comprise sequences of amino acids in peptide linkage which are arrayed either in an ⁇ -helix, a right-handed spiral, or alternatively, in a pleated sheet ⁇ -conformation. In each of these arrays the amino acids of neighboring polypeptide strands are held in place by intramolecular hydrogen bonds. Collagen, by comparison, is made up of three polypeptide chains comprising repeating amino acid trimers. These chains are arrayed in three extended left handed spirals of about three residues per turn, the polyproline II-like chains (Rich, A. et al. (1955) Nature 176:915).
  • the polyproline II-like chains of collagen are arranged in a parallel direction and intertwined to adopt a supercoiled, or coiled coil, right-handed triple helix conformation (Bella, J. et al. (1994) Science 266:75-81) that is also characteristic of collagen.
  • the chains that make up the collagen triple helix can be homotrimeric, that is, made up of identical repeating amino acid trimers, or they can be heterotrimeric, made up of chains of different amino acid trimers.
  • Mimicry of natural collagen structures has been directed to enhancing their biostability by inserting unnatural residues into the peptide sequences.
  • many unnatural proline analogs and other unnatural imino acid residues have been used to replace the frequently occurring proline residue in the peptide sequences.
  • incorporation of such residues such as the lower homologue of proline, azetidine-2-carboxylic acid (Aze), has been found to destabilize the triple helical structure of collagen or to prevent its formation (Zagari, A. et al. (1994) Biopolymers, 34:51-60).
  • Peptoid residues are a new class of unnatural imino acids (Simon, R. J. et al. (1992) Proc. Natl. Acad.Sci. USA 89:9367-9371) containing N-substituted glycine residues wherein the substituents on the nitrogen atom are the ⁇ -position side chains of amino acids. Because they are amino acids that do not occur in nature, peptoid residues or peptides containing peptoid residues have higher resistance to enzymatic attacks. In recent years, peptoid residues have been widely used in the design and synthesis of drugs and other peptide related biomaterials.
  • Template directed synthesis the interaction of one molecule with an assembly of atoms to induce a preferred molecular architecture, is known in nature.
  • the replication of DNA involves a templated synthesis of daughter polynucleotides from progenitor molecules having the same tertiary structure.
  • a template-assembling approach has been widely used for the design and synthesis of protein analogs with high molecular weights.
  • Molecular templates have been used in chemical synthesis to fix peptide loops and to induce the ⁇ -helix and ⁇ -turn structures of polypeptides.
  • Kelly, T. R. et al. (1990) J. Amer. Chem. Soc. 112:8024-8034 have reported use of a linear template to form ⁇ -structures.
  • Muller discloses anthracene-type tricyclic structures that can bridge two antiparallel peptide ⁇ -strands or induce ⁇ -turns. Muller also discloses that Kemp triacid condensed with glycine or alanine can act as a templates for inducing ⁇ -helicity of an attached polypeptide (Muller, K. et al. (1993) Chap. 33 in Perspectives in Medicinal Chemistry, B. Testa et al., eds, verlag, Basel). Ghadiri, M. et al. (1993) Angew. Chem. Int. Ed. Engl. 32:1594-1597 have prepared a polypeptide 3- ⁇ -helix bundle containing a ruthenium metal bipyridyl complex.
  • the synthetic material In order to prepare synthetic collagen that has the properties of native collagen, the synthetic material must mimic collagen in tertiary as well as primary and secondary structure.
  • a synthetic collagen-like material comprising chains of repeating trimeric amino acid building blocks, in which about 30% of the amino acids are glycine, and at least about 10% are proline or hydroxyproline, the improvement comprising the incorporation, at least in part, of peptoid residues in said repeating trimeric building blocks and formation of a peptoid residue-containing triple helix from the chains assembled from those peptoid residue containing building blocks.
  • the synthetic collagen-like material comprises chains of repeating trimeric building blocks in which the trimeric sequences are made up of a peptoid residue and two amino acid residues.
  • 30% of the residues of the trimeric sequences are glycine amino acid residues, and at least about 10% are proline or hydroxyproline amino acid residues, and at least 10% are peptoid residues.
  • These trimeric sequences each made up of two amino acid residues and one peptoid residue, are incorporated in peptoid residue-containing amino acid chains which are then formed into a peptoid residue-containing triple helix.
  • the synthetic collagen material comprises chains of repeating building blocks which are either tripeptide sequences, trimeric dipeptide-peptoid residue sequences or peptide-peptoid residue-peptide sequences selected from the group consisting of Gly-Xp-Pro; Gly-Pro-Yp; Gly-Pro-Hyp; and Gly-Pro-Pro; or combinations thereof, wherein Xp and Yp are peptoid residues, and the chains have a triple helix conformation similar to that of collagen.
  • the synthetic collagen material comprises amino acid chains made up of repeating trimeric building blocks selected from the group consisting of Gly-Xp-Pro and Gly-Pro-Yp; or combinations thereof.
  • the chains can comprise Gly-Pro-Pro and Gly-Pro-Hyp trimers.
  • the invention includes amino acid chains having terminal blocking groups, wherein the chains have the formula
  • Xp and Yp are peptoid residues; and optionally at least one tripeptide selected from the group consisting of
  • X is selected from the group consisting of linear, branched, saturated or unsaturated aliphatic acids, aromatic carboxylic acids, and aralkyl carboxylic acids and is linked to the N-terminal amino acid of the polypeptide by an amido linkage
  • Y is selected from the group consisting of ammonia, linear, branched, saturated or unsaturated aliphatic amines, aromatic amines, and aralkyl amines and Y is linked to the C-terminal end of the polypeptide by an amido linkage.
  • the peptoid residue of the repeating trimeric sequence is selected from the group consisting of N-substituted peptoid isomers of glycine, valine, leucine, isoleucine, glutamine, lysine, phenylalanine, and aspartic acid residues.
  • the repeating peptoid-residue containing triplet is of the formula Gly-Pro-Nleu or Gly-Nleu-Pro.
  • the invention also includes amino acid chains comprising repeating Gly-Pro-Hyp trimeric sequences or the amino acid chains comprising repeating trimeric sequences containing peptoid residues wherein the repulsive charges on the ends of the chains that oppose helicity are removed by acetylation at the N-terminal and amidation at the C-terminal.
  • the invention also provides template-bound amino acid chains of repeating peptide-peptoid residue trimers of the formula TP-[A-(Gly-Xp-Pro) n --NH 2 ] 3 , TP-[A-(Gly-Pro-Yp) n --NH 2 ] 3 or combinations thereof, wherein TP is a template molecule capable of inducing triple helical folding of the polypeptide chains, having three regularly spaced functional groups, each of which groups is covalently bound to a repeating peptide-peptoid residue chain; A is an optional multifunctional spacer molecule; Xp and Yp are peptoid residues; and n ⁇ 3.
  • template-bound polymers of repeating peptide trimers of the formula TP-[A-(Gly-Pro-Hyp) n --NH 2 ] 3 The amino acid chains attached to the templates can comprise combinations of tripeptides and trimeric sequences made up of a peptoid residue and two peptide residues.
  • the template molecule is the Kemp triacid (KTA) (cis, cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid), or trimesic acid (TMA) (1,3,5-benzenetricarboxylic acid);
  • KTA Kemp triacid
  • TMA trimesic acid
  • A is a spacer selected from the group consisting of Gly (--NH--CH 2 --COO--) and 6-aminohexanoic acid (--NH 2 --(CH 2 ) 5 --COO--)
  • Xp and Yp are selected from the group consisting of Nleu (add other peptoids).
  • KTA is Kemp triacid (cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid); Aha is 6-aminohexanoic acid (--NH 2 --(CH 2 ) 5 --COO--); and n is ⁇ 1.
  • Xp and Yp are peptoid residues; TMA is trimesic acid (1,3,5-benzenetricarboxylic acid); and n is ⁇ 1
  • Xp is selected from the group consisting of N-substituted glycines and other peptoid residues.
  • the invention also provides a process for inducing triple helical folding of three amino acid chains comprising the steps of (a) covalently attaching the N-terminus of one amino acid chain to each of three functional groups of a helix-inducing template molecule, either directly or through a multifunctional spacer molecule; (b) permitting the template-assembled amino acid chains to assume a stable triple helical conformation.
  • One embodiment of this aspect of the invention comprises a preliminary step of assembling the three amino acid chains by a solid phase segment condensation method on a resin support.
  • An associated process comprises attaching the N-termini of resin-bound assembled amino acid chains to the functional groups of the template, optionally through a spacer linked to the template. Alternatively, amino acids are released from the resin following assembly and the N-termini of assembled free amino acid chains are then attached to the functional groups of the template molecule in solution.
  • the amino acid chains can comprise trimeric sequences selected from the group consisting of Gly-Xp-Pro; Gly-Pro-Yp; Gly-Pro-Hyp; and Gly-Pro-Pro; or combinations thereof, wherein Xp and Yp are peptoid residues.
  • Helically arrayed collagen-like polymers according to the invention can also comprise amino acid chains of repeating trimeric sequences of the formula (Gly-Xp-Pro) n -; or (Gly-Pro-Yp) n or combinations thereof, wherein Xp and Yp are peptoid residues, and n ⁇ 3.
  • the collagen-like polymers can comprise repeating peptide trimers.
  • the repeating peptide trimers are selected from the group consisting of Gly-Pro-Hyp, Gly-Pro-Pro or combinations thereof.
  • the invention further provides synthetic collagen materials comprising the helically arrayed three amino acid chains prepared by the processes disclosed.
  • FIG. 1 depicts the Kemp Triacid template structure
  • FIG. 1(a) shows the Kemp Triacid molecule in a chair conformation in which its three carboxyl functional groups, can be coupled to the N-termini of the three amino acid chains;
  • FIG. 1(b) shows three amino acid chains of the repeated sequence (Gly-Pro-Hyp) 3 attached to the three carboxyl functional groups of a Kemp triacid molecule through glycyl spacers.
  • FIG. 2 is a circular dicroism (CD) spectrum of the repeated dipeptide-peptoid residue sequence Ac-(Gly-Pro-Nleu) 9 --NH 2 in water at 20° C., 0.2 mg/ml.
  • FIG. 3 is a circular dicroism (CD) spectrum of template-bound repeated dipeptide-peptoid residue sequences KTA-[Gly-(Gly-Pro-Nleu) 6 --NH 2 ] 3 .
  • FIG. 4 shows a comparison of melting curves of template bound chains of repeating trimers containing peptoid residues: KTA-[Aha-(Gly-Pro-Nleu) 6 --NH 2 ] 3 ; TMA-[Gly-(Gly-Pro-Nleu) 6 --NH 2 ] 3 ; KTA-[Gly-(Gly-Pro-Nleu) 6 --NH 2 ] 3 .
  • collagen-like A structure comprising ⁇ -amino acids arranged in a triple helix of polypeptide chains characteristic of collagen.
  • Each polypeptide chain has the secondary structure of a right-handed spiral, the polyproline II-like chains; and the three chains are coiled in a left-handed helical array.
  • KTA Kemp triacid
  • monodisperse for polymers and biopolymers refers to structures made up of single molecules of the uniform size or sequences of the same length and having a uniform molecular weight.
  • polydisperse for polymers and biopolymers refers to polymerized monomers or suitable building blocks. In the polymerization process, chain elongation leads to polymers with different chain lengths.
  • the polymer molecules are said to be polydisperse, that is having a distribution of sizes or lengths and molecular weights.
  • peptide or peptide residue A member of an oligomer of amino acids attached to each other by peptide bonds.
  • peptoid residue An isomeric N-substituted glycine, wherein the side chain of an ⁇ -amino acid is attached to the amino nitrogen instead of to the ⁇ -carbon of that molecule, as a member of an oligomer of amino acids attached to each other by peptide bonds.
  • peptoids Oligomers or polymers of N-substituted glycines.
  • polyproline II-like The characteristic helical conformation of a single chain collagen polypeptide comprising a left-handed extended helical coil.
  • the peptide bonds have a trans conformation.
  • Rpn An index of triple helicity which is the ratio of the intensity of the positive and negative peaks of the circular dicroism spectrum of a collagen-like assembly.
  • Gly glycine (--NH--CH 2 --COO--) and Aha: 6-aminohexanoic acid (--NH 2 --(CH 2 ) 5 --COO--).
  • TASP Template-Assembled Synthetic Proteins are artificial proteins having a predetermined three-dimensional structure formed by a process of fixing secondary-structure-forming peptide blocks on a tailor-made template molecule which directs the peptides into a packing arrangement.
  • a molecular tool typically a multifunctional molecule, that can direct the formation of a preferred molecular architecture or organization from attached chains that have the potential to assemble in a number of ways, including ordered helical arrays.
  • trimer sequence can be a tripeptide, a dipeptide attached to a peptoid residue, or a peptide-peptoid-peptide residue sequence.
  • trimesic acid (1,3,5-benzenetricarboxylic acid)
  • the peptoid residue content of the collagen-like material can be minimal.
  • peptoid residues can replace all of the proline and hydroxyproline in a typical collagen amino acid composition.
  • the collagen-like material can comprise at least about 30% glycine; and at least about 10% peptoid residue substituted for proline or hydroxyproline.
  • Remaining amino acids can those that occur in native collagen as described in Bhatnagar, R. and R. Rapaka, cited above.
  • Materials of the invention can also include collagen-like material wherein a peptoid residue occurs in every amino acid triplet.
  • helix-inducing templates can aid in assembling collagen-like polytripeptides and poly(peptide-peptoid residues) into a triple helical conformation characteristic of natural collagen.
  • triple helix formation can be demonstrated as in the Examples.
  • Peptoid residues are N-substituted glycines, wherein the side chain of an amino acid has been moved from the ⁇ -carbon to the ⁇ -nitrogen.
  • These isomers of amino acids are named as N-amino acids, that is, as Nleu, Nval, and Nlys for example.
  • Preferred peptoid residues for use in the invention are those wherein the N-substituents are the hydrophobic non-polar side-chain groups of the amino acids or their analogues, for example the isopropyl group of valine (Nval), the isobutyl groups of leucine (Nleu) or isoleucine (Nile), the propyl group of norvaline, the butyl group of norleucine.
  • Other useful peptoid residues are N-glutamic acid (Nglu), N-lysine (Nlys).
  • a peptoid residue, N-isobutylglycine (Nleu) was used as a proline surrogate.
  • collagen-like polymers comprising N-substituted peptoid residues having side-chain groupings that are similar to peptide residues are comparable to native collagen sequences and are believed to be more compatible with natural proteins than other unnatural imino acid residues.
  • Biophysical and biochemical studies indicate that unlike other proline analogs and unnatural imino acids, such peptoid residues, when incorporated into collagen-like sequences, are indeed compatible with collagen triple helical structures.
  • substituents attached to the nitrogen of the peptoid residues can be any group, for example, an aromatic or aralkyl group or a linear or branched alkyl group of at least 2 carbon atoms.
  • the substituents can also be hydrophilic or hydrophobic, and may comprise functional groups.
  • N-methyl glycine (sarcosine) wherein the N-substitutent, the methyl group of alanine, is the least hydrophobic and least bulky amino acid side group, does not provide a peptoid residue-containing chain that assembles satisfactorily into a triple helix.
  • a peptoid residue for example, N-substituted glycine
  • the reaction can be carried out in solution by several synthetic routes, for example, reductive amination of a side chain amine with glyoxylic acid as disclosed by Rogers, T. et al. (1986) Chem. Abstr. 105:226986z; or as disclosed by Buckus, P. (1964) Chem. Abstr. 61:5511b, alkylation of a side chain with haloacetic acid or, for the Ngln peptoid residue (related to glutamine).
  • Peptoid residues of the N-substituted glycine structure according to the invention are accordingly prepared by the alkylation of an alkyl- or isoalkylamine with ethyl bromoacetate. These peptoid residues are also prepared by reductive amination of C-terminal protected glycine with an alkylaldehyde. Synthesis of the peptoid residues by the alkylation and reductive amination methods is exemplified in Example 4. Both routes give high yields.
  • the single-chain structures which can form collagen-like structures according to one aspect of the invention are composed of polymers of collagen-like tripeptides each of which includes one peptoid residue.
  • Preferred peptide-peptoid residue trimers have the formula Gly-Xp-Pro or Gly-Pro-Yp wherein Xp and Yp are peptoid residues.
  • These trimeric building blocks are prepared by coupling terminal protected amino acids and peptoid residues in solution using a stepwise method.
  • the p-nitrophenol active ester Boc-Gly-ONp is prepared first and then allowed to react with proline in dimethylformamide (DMF) with a small amount of water, using triethylamine (TEA) as the catalyst.
  • DMF dimethylformamide
  • TAA triethylamine
  • the N-alkylglycine ester (Yp-OEt) is coupled to this dipeptide-free acid in DMF with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydrobenzotriazol (HOEt) as the coupling reagents to obtain the tri(peptide-peptoid residue) Boc-Gly-Pro-Yp-OEt.
  • the terminal ethyl group is removed by hydrolysis to obtain the building block tripeptoid-free acid Boc-Gly-Pro-Yp-OH.
  • the Gly-Xp-Pro peptide-peptoid species is prepared in a similar manner.
  • Boc-Gly-OH is also coupled to Xp-OEt in DMF using benzotriazolyloxytris (dimethylamino)phosphonium-hexafluorophosphate (BOP) as the coupling reagent.
  • BOP benzotriazolyloxytris (dimethylamino)phosphonium-hexafluorophosphate
  • the resulting dipeptide free acid Boc-Gly-Xp-OH is coupled to Pro-OBz using EDC and HOBt as the cooling reagents to obtain the peptoid residue-containing trimer Boc-Gly-Xp-Pro-OBz.
  • the benzyl protective group is removed by hydrogenation using Pd/C as the catalyst to obtain the trimeric building block Boc-Gly-Xp-Pro-OH.
  • trimer products (Gly-Pro-Yp-NH 2 , Ac-Gly-Pro-Yp-NH 2 , Ac-Gly-Pro-Yp-NHCH 3 Gly-Xp-Pro-NH 2 , Ac-Gly-Xp-Pro-NHCH 3 ) and (Gly-Pro-Yp) 2 --NH 2 can be prepared by peptide synthesis methods in solution.
  • Collagen-like chains comprising building blocks of repeating trimeric peptoid residue-containing sequences have been prepared.
  • preferred single chains that can form collagen analogs composed of the Gly-Pro-Nleu and Gly-Nleu-Pro repeating sequences incorporating the Nleu peptoid residue.
  • Monodisperse sequences of trimers containing the peptoid residue were prepared in order to compare the triple helical propensities of the monodisperse sequences having triplet repeats less than 10, composed of the Gly-Pro-Nleu or Gly-Nleu-Pro sequences with that of (Gly-Pro-Leu) 10 which has already been shown to be unable to form collagen-like triple helical arrays under all conditions attempted (Scatturin, A. et al. (1975) Int. J. Peptide and Protein Research 7:425-435). This comparison demonstrates the advantage of the peptoid residues over amino acid residues in triple helix formation.
  • the trimeric building blocks Boc-Gly-Pro-Yp-OH and Boc-Gly-Xp-Pro-OH can be synthesized in solution by standard peptide stepwise coupling.
  • a solution segment condensation method was used to prepare (Gly-Pro-Yp) 2 --NH 2 .
  • Boc-Gly-Pro-Yp-OH was coupled to Gly-Pro-Yp-NH 2 in DMF using BOP as the coupling reagent to get Boc-(Gly-Pro-Yp) 2 --NH 2 .
  • the Boc group was removed to obtain the N-terminal free amine trimer (Gly-Pro-Yp) 2 --NH 2 . All these small trimeric sequences prepared in solution were also purified by RP-HPLC.
  • the coupling reagents used in the solid phase synthesis included diisopropylcarbodiimide (DIC) and HOBt; the solvent used in coupling reactions was 25% DMF in DCM. A solution of TFA in DCM (30%) was used to remove the Boc group after each coupling, and a solution of 10% TEA in DCM was used for neutralization. The Kaiser ninhydrin test was used to monitor each coupling reaction. From 1.2 to 1.5 equivalents of the preferred building block trimer Boc-Gly-Pro-Yp-OH were used together with about 2 equivalents of the coupling reagents DIC and HOBt at each coupling, and the peptide bond formation normally took 4-8 hours.
  • Natural collagen and collagen-like polytripeptide sequences in the N-terminal free amine and C-terminal free acid are able spontaneously to adopt a triple helical conformation when the chains are very long.
  • cation formation on the N-termini and anion formation on the C-termini can introduce negative "end effects" that induce repulsive forces between chains and interfere with helix formation.
  • (Gly-Pro-Hyp) 10 --OH spontaneously adopts a triple helix conformation at room temperature (Venugopal, M. et al. (1994) Biochemistry 33:7948-7956); however (Gly-Pro-Hyp) 5 does not maintain helicity above 5° C.
  • Helix formation of shorter chains can be accomplished by N-terminal acetylation and C-terminal amidation which removes the described end effects.
  • a suitable N-terminal acetylating agent is any molecule having a carboxyl group available for binding the terminal amine.
  • Preferred agents are linear, branched, saturated or unsaturated aliphatic acids, aromatic carboxylic acids, and aralkyl carboxylic acids.
  • a suitable C-amidation agent is any molecule having an amine group available for binding the terminal carboxyl of the chain.
  • Preferred agents are ammonia, linear, branched, saturated or unsaturated aliphatic amines, aromatic amines, and aralkyl amines.
  • Non-limiting examples of suitable C-terminal groups are amide, bipyridine, and maleimide.
  • acetic anhydride was used to acetylate the N-termini of the trimeric peptoid residue-containing amide compounds in DCM with triethylamine as the base.
  • the preparation of these products is set forth in Example 8.
  • N-terminal acetylation of single chains can be demonstrated by comparing the melting temperatures of (Gly-Pro-Hyp) 5 , (5° C. in 50% ethanol/water), and Ac-(Gly-Pro-Hyp) 5 --NH 2 , 23° C. in 50% ethanol/water). Accordingly, embodiments of this aspect of the invention include N-terminal acetylated single chain polypeptides of the general formula Ac-n, wherein n represents the number of repeating collagen-like trimers.
  • Preferred members of this group are collagen chains of the formulas Ac-(Gly-Pro-Hyp) n --NH 2 , Ac-(Gly-Xp-Hyp) n or Ac-(Gly-Hyp-Yp) n .
  • a template with three functional groups to connect the three chains covalently or non-covalently at the same end (C-terminus or N-terminus) of the peptide chains is believed to be a feasible route to nucleate and propagate collagen triple helix formation.
  • a second type of collagen-like peptoid residue-containing structure is based on analogs with terminal templates which induce triple helical conformations in these poly(peptide-peptoid residue) chains.
  • a template with functional groups is used to help the formation of a specific protein (or polypeptide) conformation.
  • Templates have been used as part of a process termed Template-assembled-Synthetic-Protein (TASP:Tuchscherer, G. and Mutter, M. (1995) Journ. Peptide Sci. 1:3-10 to mimic the ⁇ -helix and ⁇ -turn structures of polypeptides, but have not been used previously to mimic collagen-like triple helical conformations.
  • Two types of templates were used in a preferred approach according to the invention.
  • One is based on derivatives of the Kemp triacid (KTA), a cyclohexane derivative which has three carboxyl functional groups all in the axial positions (Kemp, D. and K. Petrakis (1981) J. Org. Chem. 46:5140-5143).
  • the second is based on derivatives of the trimesic acid (TMA) which has three carboxyl functional groups oriented in the plane of the benzene ring.
  • TMA trimesic acid
  • the highly constrained templates of the invention are believed to be superior to the highly flexible templates based on lysine dipeptides and other bridge structures of Roth (1980) and Fields (1993), cited above.
  • the highly constrained templates of the invention while holding the chains in parallel, can by means of the bending and flexing of the molecule frame, allow the residues of those chains to seek optimal register by movement along a path.
  • the templates of Roth and Fields by contrast, promote register of the chains by adjusting the alignment of each chain to another with a single residue increment shift.
  • Non-limiting examples of other trifunctional templates that are suitable, (although somewhat more flexible), for inducing collagen triple helix formation among collagen-like polypeptides or peptoid residue-containing chains of the invention are ⁇ -cyclodextrin, and aminotrithiol compounds.
  • Functionalized molecules having the structures of fused cyclohexanes, condensed aromatics, adamantane, or steroids are suitable as helix-inducing templates according to the invention.
  • Those skilled in the art can identify other suitable template structures.
  • the KTA templates are preferred as more compatible with collagen triple helical conformations because the three functional groups of the triacid can be coupled to the three peptide chains in collagen triple helical structures.
  • the Kemp triacid KTA cis, cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid
  • a conformationally constrained organic structure was accordingly used as a template to nucleate the triple helical folding of three polypeptide chains containing (Gly-Pro-Hyp) triplet repeats.
  • KTA prefers a chair conformation in which the three carboxyl functional groups are in the axial positions.
  • the separation between the three axial carbonyl carbon atoms is 2.5-3.5 ⁇ (Kemp, D. and K. Petrakis, 1981), while the distances between symmetry-related carbonyl carbon atoms bound to the Gly-NH in triple helical sequences are always greater than 4.
  • a spacer for example, a glycine residue, can be inserted between each peptide chain and each carboxyl group on the template molecule, in this example KTA.
  • the three polypeptide or peptoid residue-containing chains are therefore preferably linked to the templates through spacer molecules; glycine residues that act as spacers are preferred.
  • a spacer junction between the template and the helix strands serves the purpose that it relieves steric hindrance among the chains.
  • the three chains are shifted by one residue.
  • the triple helix packing involves a Gly from one chain, a Pro from a second chain and a Hyp from a third chain. Because the spacer can twist and stretch to different conformations, the three peptide chains can adopt the proper shift necessary for the collagen-like triple helix array. Accordingly, when each carboxyl group of a Kemp triacid or trimesic acid is coupled to a strand of the triple helix through a flexible spacer such as glycine, the assembly allows for the proper shift and helical register.
  • the C-terminal benzylester-protected spacer residues (Gly-OBz, Aha-OBz) were first coupled to KTA and TMA in DMF by using EDC and HOBt as the coupling reagents.
  • the C-terminal benzyl groups were removed by hydrogenation in methanol using Pd/C as the catalyst to produce the preferred templates with three carboxyl groups: KTA-(Gly-OH) 3 , KTA-(Aha-OH) 3 and TMA-(Gly-OH) 3 .
  • the purity of these templates were checked by analytical HPLC profiles, and if necessary, they were further purified by performing preparatory RP-HPLC.
  • Two methods were used to attach the templates with carboxyl functional groups to the N-termini of peptide chains.
  • the peptide-peptoid residue chains were assembled first on the resin. After a specific chain length was reached, the template was coupled to the N-termini by using DIC and HOBt as the coupling reagents. The product was removed from the resin by the HF cleavage method and purified by HPLC. This method was successfully used in the preparation of template-assembled collagen-like polypeptides composed of Gly-Pro-Hyp sequences. Some of the preferred template-assembled collagen-like polypeptide-peptoid residue trimers composed of Gly-Pro-Yp and Gly-Xp-Pro sequences were also prepared by this method.
  • the templates and the peptide chains were connected through a peptide bond formation in solution.
  • the peptide-peptoid residue chains were assembled by the solid phase synthesis method and were cleaved from the resin as N-terminal free amines. These peptoid residue chains were purified by HPLC.
  • the templates and the free-amine peptide-peptoid residue chains were coupled in solution using EDC and HOBt. This method was used in the preparation of several target template-assembled repeated trimeric building blocks, which optionally contained peptoid residues.
  • the innovative design of collagen templates according to the invention is based on the observation that the KTA template is skewed compared to the triple helix axis as shown by molecular mechanics simulations. This observation has two important consequences which are relevant for the general design rationale of collagen triple helix templates.
  • the skewing mentioned above allows the template to adapt to the collagen triple helix register shift without any significant distortion of its geometry, as indicated by the KTA methylene chemical shifts which are very sensitive to conformational changes (Kemp, D. et al. 1981 J. Org. Chem. 46:5140-5143).
  • the template can therefore be highly constrained without the risk of significantly distorting the triple helical array. This means that the free energy gain in the collagen triple helix formation obtained by highly constrained templates with proper spacers is greater than that allowed by the highly flexible templates published so far (Roth, W. et al. 1980 Biopol., 19:1909-1917; Fields, C. G. et al.
  • the skewing of the template as compared to the triple helix axis eliminates the requirement that the highly constrained templates have a ternary screw symmetry with the correct triple helix chirality.
  • the collagen triple helix will instead induce the correct symmetry and transfer chirality to the template, as shown by modeling studies and by the splitting of the KTA signals in the NMR spectra acquired in conditions under which the triple helix is present.
  • the highly constrained templates can therefore be achiral and have ternary rotational symmetry. This design rationale leads to target molecules which are more easily accessible by chemical synthesis than the chiral templates with screw symmetry.
  • the compound TMA-[(Gly-Pro-Nleu) 9 --HN 2 ] 3 represents a molecule which does not possess any spacer residues between the template (TMA) and the peptide chains.
  • This compound was prepared to show the necessity of spacers and a preferred conformation of the template with respect to the triple helix formation, especially when the peptide chains are long enough to compensate for the structural effects of an unpreferred template.
  • the TMA was activated to the acid chloride and coupled to the N-terminal of the peptide-peptoid residue (Gly-Pro-Nleu) 9 --NH 2 in solution. Examples of the preparation of template-linked collagen-like polypeptides are presented in Examples 9-11.
  • the amount of the template used is crucial. To exclude the formation of by-products such as compounds with only one chain and/or two chains attached to the template, the equivalents of the template used must be controlled. In a preferred synthesis, the template was added portionwise. In between the additions, the Kaiser test was used to monitor the reaction. In this way, the coupling of the template to the polypeptide-peptoid residue chains normally took 2-3 days, and the functional group equivalents of the template used were generally below 0.9.
  • Collagen-like and polyproline II-like structures exhibit unique CD spectra in solutions (Sakakibara, S. et al. (1968)). These spectra are characterized by a large negative peak around 200 nm and a small positive peak around 217-227 nm. These features have been used as a basis to establish the presence of a polyproline II-like or collagen-like triple helical structure in solution for natural and synthetic peptides (Inouye, K. et al. (1982)). However, these CD spectral shapes and peak positions do not show whether polyproline II-like peptide chains have associated to triple helical structures. Therefore, they cannot be used conclusively, although these parameters are necessary, to establish the presence of a collagen-like triple helical conformation.
  • a criterion for evaluating the occurrence and extent of triple helicity in peptide/peptoid residue containing molecules is provided by the peak intensities and the wavelength of the minimum and maxima of the CD spectra of these molecules.
  • the intensities of both the positive and negative peaks increase as the chain length increases and decrease when the temperature increases. Because the peak intensities and their position depend on many factors, they cannot be used unambiguously to distinguish triple helicity from other structural arrays such as a polyproline II type structure. We have found, however, that an important parameter related to the peak intensities can be useful in establishing the presence of a triple helical array in solution. This parameter is the ratio of the intensities of the positive to negative peak. This parameter is termed Rpn.
  • Thermal melting measurements describe the change in a physical property of a collagen-like helix, for example the change of specific rotation or UV absorption with temperature. This provides another indication of triple helix formation and stability. Helix formation is indicated by a transition in the rate of change of property with temperature, indicated by an inflection in the melting curve. The transition temperature indicates that temperature above which the helix is denatured. Melting curves for template-bound chains of Gly-Pro-Nleu chains are shown in FIG. 4.
  • a template can significantly facilitate triple helix formation by allowing a shorter chain to form a triple helical structure and stabilize the triple helical conformations.
  • the system KTA-[Gly-(Gly-Pro-Hyp) 3 --NH 2 ] 3 forms a triple helix, and represents the shortest chain collagen-like triple helix yet reported. Results show that both [KTA-[Gly-(Gly-Pro-Hyp) 5 --NH 2 ] 3 and [KTA-[Gly-(Gly-Pro-Hyp) 6 --NH 2 ] 3 form triple helical structures which can be denatured only above 70° C. in water.
  • the effect of the template-spacer is even more dramatic for short chains made up of the Gly-Pro-Hyp trimer.
  • [KTA-[Gly-(Gly-Pro-Hyp) 3 --NH 3 ] 3 is triple helical in water at room temperature, while Ac-(Gly-Pro-Hyp) 3 --NH 2 does not adopt a triple helical structure even in a more favorable solvent system, ethylene glycol:water (v/v 2:1), and at lower temperatures.
  • This invention similarly includes use of the other peptide-peptoid residue building blocks to form novel collagen-like structures.
  • co(peptide-peptoid residue) sequences can be included by specific placements of specific building blocks in the collagen-like structures.
  • the sequences of the invention can be analogous to native collagen, comprising either homotrimeric or heterotrimeric chains.
  • Heterotrimeric chains can comprise mixtures of tripeptides and dipeptide-peptoid residue trimers having, for example, the following structure:
  • j,k,l, and m designate the number of repeats for that trimer and the sum of j to m is n, the number of repeating trimeric units.
  • n the number of repeating trimeric units.
  • trimers there are at least three trimers in the chain (n ⁇ 3). The trimers can occur in any order.
  • triple helices of the invention can similarly comprise chains of the same sequence of trimers or a different sequence. In any particular case, triple helix formation can be demonstrated as in the Examples.
  • MILLENNIUM 2010 system consisted of a Waters 715 Ultra WISP sample processor, a Waters TM 996 photodiode array detector, two Waters 510 pumps and a NEC PowerMate 486/33I computer.
  • Solvents used in HPLC were solvent A: H2O with or without 0.1% TFA, solvent B: acetonitrile with or without 0.1% TFA.
  • the flow rate was 10 ml/min. for preparatory column, 4 ml/min. for semipreparatory column, and 1.0-1.2 ml/min. for analytical column.
  • NMR NMR spectroscopy
  • Circular dichroism Measurements were carried out on a modified Cary-61 Spectropolarimeter. CD spectra were obtained with a 0.02 cm cell, by signal-averaging 10 scans. The wavelength range was set between 185-300 nm, the scan speed was 1.0 nm per second. The sample was measured at a concentration 0.2 mg/ml. To allow for proper equilibration of triple helix formation, the solution was kept in a refrigerator ( ⁇ 4° C.) for at least 24 hours prior to each experiment and another 2 hours at the specified temperature before acquiring data.
  • UV The ultraviolet (UV) melting curve was carried out on a Cary-1E UV Spectrometer.
  • the sample concentration was 0.04 mg/ml and was prepared from the 0.2 mg/ml solution for the CD measurements.
  • the solution was kept in a refrigerator (4° C.) at least 24 hours before experiment.
  • To perform the melting experiments the sample was equilibrated one hour at the starting temperature.
  • the heating rate was 0.2 degree per minute.
  • the measuring wavelength was set at 223 nm.
  • MS Mass spectra were obtained at UC Riverside and the Scripps Research Institute. Fast atom bombardment (FAB), electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) methods were used to verify the structures of the products.
  • FAB Fast atom bombardment
  • ESI electrospray ionization
  • MALDI matrix-assisted laser desorption ionization
  • Boc-Gly-OH 35 g, 0.2 mol.
  • Tos-Pro-OBz 75.5 g, 0.2 mol.
  • HOBt 30 g, 0.22 mol.
  • the solution was cooled on an ice water bath and TEA (35 ml, 0.25 mol.) was added slowly. After 5 minutes, EDC (40 g, 0.21 mol.) was added. After stirring the solution at 0° C. for one hour, the bath was removed and the solution was stirred at room temperature overnight. The DMF was removed under reduced pressure. The remaining mixture was decanted into 800 ml ethyl acetate.
  • the Boc-Gly-Pro-OBz (19.4 g, 0.054 mol.) was dissolved in a mixture of 100 ml H 2 O and 100 ml THF. After cooling the solution to 0° C., KOH (4.8 g, 0.085 mol.) in 50 ml H 2 O was added slowly. The solution was stirred for one hour. Then, the THF was removed under reduced pressure and the aqueous solution was extracted with ethyl acetate to remove the benzyl alcohol. The resulting aqueous phase was covered by 200 ml ethyl acetate and was acidified at 0° C. to about pH 2 by addition of concentrated HCl slowly (vigorously swirling the solution at the same time).
  • Boc-Gly-Pro-OH (2.8 g, 0.01 mol.) and p-nitrophenol (1.7 g, 0.012 mol.) were dissolved in DCM (100 ml) and DCC (2.1 g, 0.01 mol.) was added to the solution by portions while stirring the solution. A precipitate (dicyclohexylurea, DCU) soon formed. After stirring the solution at room temperature for 3.5 hours, TLC analysis (developing solvents:ethyl acetate/hexane, 3:1) showed that all the starting material Boc-Gly-Pro-OH was consumed. The Rf for the product Boc-Gly-Pro-ONp is 0.45 in this solvent system. DCU was removed by filtration and DCM was removed under reduced pressure.
  • the aqueous solution was extracted with ethyl acetate 2 ⁇ 100 ml to remove unreacted benzyl chloride. Then, the aqueous solution was covered by 200 ml ethyl acetate and was acidified at 0° C. to pH 3 by addition of concentrated HCl slowly (vigorously swirling the solution at the same time). The products were extracted from the aqueous solution with ethyl acetate 2 ⁇ 200 ml. Two compounds were found in the organic layer by TLC analysis (developing solvents: chloroform/methanol/acetic acid (CMA), 85:15:3). They were verified by NMR to be the expected product Boc-Hyp(OBz)--OH (Rf, 0.36).
  • Boc-Gly-Pro-ONp (0.01 ml) was added and the resulting solution was stirred for one hour. The bath was removed and the stirring was continued overnight. The solvents were distilled under reduced pressure and the remaining mixture was poured into 50 ml saturated NaHCO 3 . The aqueous solution was extracted with ethyl acetate 2 ⁇ 50 ml to remove organic impurity. The remaining aqueous solution was covered by 100 ml ethyl acetate and was acidified at 0° C. to about pH 2 by addition of concentrated HCl slowly (swirling the solution vigorously at the same time). The product was extracted from the aqueous solution with ethyl acetate 2 ⁇ 150 ml.
  • Boc-Gly-Pro-Hyp-(OBz)-MBHA (0.3 mmol. based on the resin substitution) was prepared following the general solid phase segment condensation procedure described in the section on General Information.
  • the Boc protecting group was removed using a solution of 15 ml 30% TFA in DCM, 1.0 ml anisol was added as scavenger.
  • the resin was washed with 20 ml DCM, 20 ml methanol, 20 ml DCM and followed by 2 ⁇ 15 ml 10% TEA in DCM.
  • the n-termini were acetylated using 0.5 ml acetic anhydride dissolved in 15 ml DCM with 5% TEA to give Ac-Gly-Pro-Hyp(OBz)-MBHA.
  • Boc-Gly-Pro-Hyp(OBz)-MBHA (0.3 mmol based on resin substitution level) was prepared following the general solid phase synthesis procedure described in the section on General Information.
  • the Boc group was removed using a solution of 30% TFA in DCM and 1.0 ml anisol was added as scavenger.
  • the resin was washed with DCM, methanol, 10% TEA in DCM and DCM to give Gly-Pro-Hyp(OBz)-M3HA.
  • KTA-(Gly-OH) 3 35 mg, 0.08 mmol
  • HOBt 50 mg
  • Boc-Gly-OH (32.6 g, 0.186 mol.) and p-nitrophenol (27.8 g, 0.20 mol.) were dissolved in 300 ml dichloromethane (DCM) and the solution was chilled to 0° C. on a water-ice bath.
  • DCM dichloromethane
  • DCC dicyclohexylcarbodiimide
  • DCU dicyclohexylurea
  • Boc-Gly-ONp 50 g, 0.17 mol.
  • proline 20.7 g, 0.175 mol.
  • 300 ml DMF 20 ml TEA and 30 ml water were added while stirring the solution. The stirring was continued overnight and TLC analysis showed no more starting materials.
  • the solvents were removed under reduced pressure and the resulting mixture was dissolved in 250 ml saturated Na 2 CO 3 .
  • Boc-Gly-Pro-OH (3.85 g, 0.014 mol), the HCl salt of Nleu-OEt (2.76 g, 0.014 mol) and HOBt (2.8 g, 0.02 mol.) were dissolved in 100 ml DMF and the solution was chilled to 0° C. on a water-ice bath.
  • TEA (2.8 ml, 0.02 mol.) was added to the solution.
  • EDC 4.0 g, 0.021 mol.
  • the stirring was continued overnight.
  • the DMF was removed under reduced pressure and the resulting mixture was poured into 200 ml ethyl acetate.
  • the organic layer was washed by H 2 O 2 ⁇ 30 ml, saturated NaHCO 3 3 ⁇ 10 ml, brine 2 ⁇ 10 ml, saturated NaHSO 4 3 ⁇ 15 ml and brine again until the pH value of the brine layer was approximately 7.
  • the ethyl acetate solution was dried using Na 2 SO 4 and the solvent was removed by distillation under reduced pressure.
  • Column chromatography (elution solvents: E/H, 2:1) was performed to obtain the pure product (yellowish oil, 4.7 g, 81%).
  • Boc-Gly-Pro-Nleu-OEt (4.3 g, 0.0104 mol.) was dissolved in 80 ml THF/H 2 O (v/v, 1:1) and the solution was chilled to 0° C. on a water-ice bath.
  • KOH 1.1 g dissolved in 20 ml H 2 O
  • the THF was removed under reduced pressure.
  • the aqueous solution was extracted by ethyl acetate 2 ⁇ 50 ml. Then, the aqueous layer was chilled to 0° C.
  • Boc-Gly-OH (4.4 g, 0.025 mol.) and Nleu-OEt (3.2 g, 0.02 mol.) were dissolved in 100 ml DMF and the solution was chilled to 0° C. on a water-ice bath. BOP (11 g, 0.025 mol.) was added to the solution. Triethylamine was used to adjust the pH of the solution to about 9. The solution was stirred overnight. The DMF was removed under reduced pressure and the resulting mixture was poured into 300 ml ethyl acetate. The ethyl acetate solution was washed by H 2 O, saturated NaHCO 3 , brine, saturated Na 2 SO 4 and brine again.
  • Boc-Gly-Nleu-OEt (28.8 g, 0.094 mol.) was dissolved in a mixture of 150 ml H 2 O and 150 ml THF.
  • KOH (11.9 g, 0.18 mol.) dissolved in 50 ml H 2 O was added while stirring the solution. After one hour, The THF was removed under reduced pressure.
  • the product was extracted from the aqueous layer by ethyl acetate 3 ⁇ 100 ml. The organic layers were combined, washed by brine and dried by Na 2 SO 4 .
  • Boc-Gly-Nleu-OH (12.5 g, 0.043 mol.), HCl•Pro-OBz (11 g, 0.046 mol.) and HOBt (6.0 g, 0.045 mol.) were dissolved in 300 ml DMF and the solution was chilled to 0° C.
  • Triethylamine (12 ml) was added slowly while stirring the solution.
  • EDC 8.5 g, 0.043 mol.
  • the DMF was removed under reduced pressure and the resulting mixture was poured into 350 ml ethyl acetate.
  • Boc-Gly-Pro-Nleu-OH (4.1 g, 0.0106 mol.), NH 4 Cl (1.7 g, 0.032 mol.) and HOBt (1.76 g, 0.013 mol.) were dissolved in 100 ml DMF and the solution was chilled to 0° C.
  • TEA 4.5 ml was added to the solution slowly while stirring the solution.
  • EDC 2.6 g, 0.013 mol.
  • the chloroform solution was washed by H 2 O 2 ⁇ 20 ml, saturated NaHCO 3 2 ⁇ 20 ml, brine 2 ⁇ 20 ml, saturated NaHSO 4 2 ⁇ 20 ml and brine again until the pH of the brine layer was approximate 7.
  • the organic layer was dried using Na 2 SO 4 and the chloroform was distilled under reduced pressure to obtain the product (3.5 g, 84%).
  • TLC Rf 0.36 (developing solvents: CMA, 85:15:3).
  • Boc-Gly-Pro-Nleu-NH 2 (3.5 g, 0.0089 mol.) was dissolved in a solution of 30% TFA in DCM (50 ml). The solution was stirred for 40 minutes. The DCM and TFA were removed under reduced pressure, and 3 ⁇ 50 ml benzene were added and distilled under reduced pressure to remove trace TFA. The resulting mixture was dissolved in 30 ml methanol and the solution was chilled to 0° C. HCl/dioxane (4N, 10 ml) was added to the solution to transfer the product from TFA salt to HCl salt. Ethyl ether (200 ml) was added to the solution and a precipitation formed.
  • Boc-Gly-Pro-Nleu-OH (0.3 g, 0.76 mmol.) and Gly-Pro-Nleu-NH 2 (0.00075 mol.) were dissolved in 20 ml DMF and the solution was chilled to 0° C.
  • TEA 0.3 ml was added slowly while stirring the solution.
  • BOP 0.5 g, 0.0012 mol. was added to the solution and the stirring was continued overnight.
  • the DMF was removed and the resulting mixture was poured into 150 ml chloroform.
  • the chloroform solution was washed by H 2 O 2 ⁇ 10 ml, saturated NaHCO 3 2 ⁇ 10 ml, brine 2 ⁇ 10 ml, saturated NaHSO 4 2 ⁇ 10 ml and brine again until the pH of the brine layer was approximate 7.
  • the organic layer was dried using Na 2 SO 4 and the chloroform was distilled under reduced pressure to obtain the Boc-(Gly-Pro-Nleu) 2 --NH 2 .
  • This product was dissolved in a solution of 30% TFA in DCM (30 ml) and the solution was stirred at room temperature for 30 minutes. The DCM and TFA were removed under reduced pressure, and 3 ⁇ 20 ml benzene were added and distilled under reduced pressure to remove trace TFA.
  • Boc-Gly-Pro-Nleu-OH (0.77 g, 2 mmol), HClNH 2 CH 3 (0.27 g, 4 mmol.) and HOBt (0.35 g, 2.5 mmol) were dissolved in DMF and the solution was chilled to 0° C. on a water-ice bath. Triethylamine (0.7 ml) was added slowly while stirring the solution. After 5 minutes, EDC (0.46 g, 2.4 mmol.) was added to the solution and the stirring continued overnight. The DMF was removed under reduced pressure and the resulting mixture was poured into 150 ml chloroform.
  • the chloroform solution was washed by H 2 O, saturated NaHCO 3 , brine, saturated NaHSO 4 and brine again until the pH of the brine layer was approximate 7.
  • the organic layer was dried using Na 2 SO 4 and the chloroform was distilled under reduced pressure to obtain the Boc-Gly-Pro-Nleu-NHCH 3 .
  • TLC Rf 0.44 (developing solvents: CMA, 85:15:3). This product was used directly in next step without further purification and characterization.
  • 30 ml solution of 30% TFA in DCM was used to remove the Boc group. The deprotection was allowed to proceed 30 minutes. Then, the DCM and TFA was removed under reduced pressure and benzene 3 ⁇ 20 ml was added and distilled to remove trace TFA.
  • the TFA salt of Gly-Pro-Nleu-NHCH 3 was dissolved in 50 ml and the solution was chilled to 0° C. Triethylamine was added slowly to neutralize the peptide. After a while, acetic anhydride (2ml) was added and the acetylation was allowed to proceed 40 minutes. The DCM was removed and the resulting mixture was dissolved in 100 ml chloroform. The chloroform solution was washed by H 2 O, saturated NaHCO 3 , brine, saturated NaHSO 4 and brine again until the pH of the brine layer was approximately 7.
  • polypeptides composed of the Gly-Pro-Hyp and Gly-Pro-Pro sequences were prepared as references to establish the triple helical propensity of the collagen-like poly(peptide-peptoid residue) structures.
  • the Kemp triacid (KTA) (0.1, g, 0039 mol.) TOS-Gly-OBz (6.5 g, 0.019 mol.) and HOBt (2.6 g, 0.019 mol.) were dissolved in 50 ml DMF.
  • the solution was chilled to 0° C. and TEA (5 ml, 0.036 mol.) was added slowly. After stirring the solution for 5 minutes, EDC (3.7 g, 0.019 mol.) was added. The reaction continued 4 hours at room temperature.
  • the DMF was distilled under reduced pressure and the remaining mixture was poured into 300 ml ethyl acetate.
  • 6-aminohexanoic acid benzyl ester Aha-OBz 6-aminohexanoic acid (Aha-OH) (13.1 g, 0.1 mol), benzyl alcohol (100 mL), benzene (100 mL) and tolylsulfonic acid (19 g, 0.1 mol) were mixed and the mixture was refluxed for 3 h using a Dean Stark apparatus. The resulting mixture was cooled to room temperature and 500 mL ethyl ether was added. The precipitate was collected by filtration and washed using ethyl ether and dried in vacuum overnight to get a white solid product Tos-Aha-OBz (40.5 g, 98%).
  • Tos-Gly-OBz (8.4 g, 0.025 mol) was dissolved in DCM (150 mL) and the solution was chilled to 0° C. TEA (5 mL) was added slowly. After 10 minutes, trimesic acid chloride (1.4 g, 0.005 mol) was added and the solution was stirred at room temperature for 5 h. DCM was removed under reduced pressure and the resulting mixture was poured into 300 mL ethyl acetate.
  • the solid phase method (Gly-Pro-Nleu) 6 -MBHA (0.3 mmol based on the resin substitution) was obtained using the general solid phase synthesis procedures described in the General Information section.
  • KTA-(Gly-OH) 3 (30 mg, 0.07 mmol) was coupled to the N-termini of the peptide-peptoid residue chains using DIC and HOBt as the coupling reagents and DMF/DCM (v/v, 1:1) as the solvents. Two couplings were performed to ensure complete functionalization. For the first coupling, 0.035 mmol template was used and the coupling was allowed to proceed for one day. The solution was removed from the reaction vessel by filtration and the resin was washed with DCM (3 ⁇ 20 mL).
  • the mixture was filtered and another 20 mg (0.047 mmol) KTA-(Gly-OH) 3 was used (with coupling reagents DIC and HOBt) for a second coupling. This second coupling was also allowed to proceed one day.
  • the template-assembled peptide-peptoid product was removed from the resin by the HF cleavage method and was dialyzed against water in a 3500 dalton cut-off membrane tubing for 3 days. HPLC was carried out to obtain the pure proudct (30 mg, 4.4%), A product with two chains attached to the template was also obtained.
  • MALDI MS, observed (M+Na)+ 5259.
  • Analytical HPLC chromatogram, RT 14.3 min. (35-70% B, 30 min).
  • TMA-(Gly-OH) 3 (0.1 g, 0.25 mmol), HCl(Gly-Pro-Nleu-NH 2 (0.26 g, 0.85 mmol) and HOBt (0.14 g, 1.0 mmol) were dissolved in DMF (3 mL) and the solution was chilled to 0° C. TEA (0.2 mL) was added slowly while stirring the solution. After 10 minutes, EDC (0.19 g, 1.0 mmol) was added and the stirring was continued overnight at room temperature. The DMF was removed under reduced pressure and the resulting mixture was dissolved in water. Dialysis was performed in a membrane tubing with cut-off of 1000 dalton for 9 hours to remove low molecular weight impurities.
  • Boc-Gly-Nleu-Pro-OH (1.1 g, 2.85 mmol.), NH 4 Cl (0.5 g, 8.6 mmol.) and HOBt (0.5 g, 3.7 mmol.) were dissolved in DMF and the solution was chilled to 0° C. on a water-ice bath. Triethylamine (1.4 ml) was added slowly while stirring the solution. After 10 minutes, EDC (0.73 g, 3.7 mmol.) was added to the solution and the stirring continued overnight. The DMF was removed under reduced pressure and the resulting mixture was poured into 300 ml ethyl acetate.
  • the ethyl acetate solution was washed by H 2 O 2 ⁇ 10 ml, saturated NaHCO 3 2 ⁇ 10 ml, brine 2 ⁇ 10 ml, saturated NaHSO 4 2 ⁇ 10 ml and brine again until the pH of the brine layer was approximate 7.
  • the organic layer was dried using Na 2 SO 4 and the ethyl acetate was distilled under reduced pressure to obtain the product Boc-Gly-Nleu-Pro-NH 2 .
  • the Boc group of this product was removed in 30 ml solution of 30% TFA in DCM.
  • the deprotection reaction was allowed to proceed for 40 minutes.
  • the DCM and TFA was removed under reduced pressure and benzene 3 ⁇ 20 ml was added and distilled to remove trace TFA.
  • Boc-(Gly-Pro-Nleu) 3 -MBHA (0.35 mmol. based on the resin substitution) was obtained using the solid phase synthesis procedures described in the General Information section.
  • the Boc group was removed by a solution of 30% TFA in DCM. The deprotection was allowed to proceed for 30 minutes.
  • the N-termini were acetylated using acetic anhydride (2 ml) in DCM with 5% TEA. The acetylation reaction took 30 minutes.
  • HF cleavage lyophilizing was performed to get the crude product and HPLC was carried out to obtain the pure product (47%).
  • Boc-(Gly-Pro-Nleu) 3 -MBHA (0.35 mmol. based on the resin substitution) was obtained using the solid phase synthesis procedures described in the General Information section.
  • the Boc group was removed by a solution of 30% TFA in DCM. The deprotection was allowed to proceed for 30 minutes.
  • the N-termini were acetylated using acetic anhydride (2 ml) in DCM with 5% TEA. The acetylation reaction took 30 minutes.
  • lyophilizing was performed to get the crude product and HPLC was carried out to obtain the pure product (47%).
  • peptoid-containing collagen-like structures contain unnatural residues (peptoid residues) which enhance the biostability of these compounds.
  • the peptide-peptoid collagen-like structures represent a new class of novel collagen-like biomaterials.

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US20050250857A1 (en) * 2002-05-22 2005-11-10 Van Bommel Kjeld Jacobus Corne Gelling agents
US20060099270A1 (en) * 2003-05-22 2006-05-11 Applied Nanosystems B.V. Production of small particles
US20070060508A1 (en) * 2002-08-06 2007-03-15 Udo Haberl Binding molecules
US20070275897A1 (en) * 2006-05-26 2007-11-29 Raines Ronald T Collagen mimics
EP1394180B1 (de) * 2002-08-06 2008-07-16 AplaGen GmbH Synthetische Mimetika von physiologischen Bindungsmolekülen
US20090106360A1 (en) * 2007-06-08 2009-04-23 Jin Peng Content delivery method and system, network device, and mobile data service platform
US20090299034A1 (en) * 2007-08-01 2009-12-03 Mabel Alamino Cejas Collagen-related peptides
US20100021527A1 (en) * 2008-07-25 2010-01-28 Chunlin Yang Collagen-related peptides and uses thereof and hemostatic foam substrates
US8076294B2 (en) 2007-08-01 2011-12-13 Advanced Technologies And Regenerative Medicine, Llc. Collagen-related peptides and uses thereof
US20140371145A1 (en) * 2011-09-29 2014-12-18 Fujifilm Corporation Scaffold for vascular endothelial cell migration
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NL1007908C2 (nl) * 1997-12-24 1999-06-25 Fuji Photo Film Bv Zilverhalide-emulsies met recombinant collageen die geschikt zijn voor fotografische toediening alsmede de bereiding daarvan.
FR2798655B1 (fr) * 1999-09-21 2001-11-16 Oreal Composition comprenant un compose derive de cyclohexane, compose et utilisation dudit compose pour structurer une composition
EP1420806B1 (en) 2001-07-12 2013-05-01 Rikard Holmdahl Antibody detection method using triple polypeptide complexes derived from collagen ii
WO2007143121A2 (en) 2006-06-01 2007-12-13 President And Fellows Of Harvard College Purification of a bivalently active antibody using a non-chromatographic method
WO2008140595A2 (en) * 2006-12-01 2008-11-20 President And Fellows Of Harvard College Synthetic trivalent haptens, complexes thereof, and uses therefor
EP2418284A1 (en) * 2010-08-13 2012-02-15 ERA Biotech, S.A. Protein body-inducing polypeptide sequences
JP6051619B2 (ja) * 2012-06-29 2016-12-27 Jnc株式会社 コラーゲン様ポリペプチドの製造方法
CN107857813A (zh) * 2017-12-05 2018-03-30 陕西慧康生物科技有限责任公司 一种三肽‑29的液相合成方法

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